Insulin-like growth factor I regulates type I procollagen messenger ribonucleic acid steady state levels in bone of rats

RNA extracted from parietal bones of rats was analyzed for the abundance of type I procollagen mRNA by a dot hybridization assay using specific cDNA probes. Hypophysectomized rats, deficient in IGF I, have 4.1-fold lower steady state levels of mRNA encoding the alpha 1 chain of type I collagen when compared to normal rats of the same weight (120 g). Administration of recombinant human insulin-like growth factor I (IGF I) for 6 days restores bone collagen mRNA levels to normal, and a single sc injection of 100 micrograms IGF I results in a 1.9-fold increase of both alpha 1(I) and alpha 2 (I)chain mRNAs after 8 h. Studies with calvaria cells in primary culture and with a bone-derived cell line, PyMS, yielded similar results: IGF I treatment of these osteoblast-like cells for 24 h increased steady state levels of type I procollagen mRNAs. Thus, IGF I appears to contribute to osteoblast mRNA expression for both chains of type I collagen.     

Rapid alteration of pancreatic proinsulin and preproinsulin messenger ribonucleic acid in rats treated with cyproheptadine

Cyproheptadine (CPH) appears to be unique among islet beta-cell toxicants by virtue of its ability to rapidly and reversibly inhibit insulin biosynthesis in rats. These studies examined the mechanism of CPH-induced insulin depletion by determining the time course for CPH-induced changes in pancreatic preproinsulin mRNA, proinsulin, and insulin levels. A single oral dose of CPH decreased proinsulin levels to 35% of the control value by 3 h. Proinsulin stayed depressed for up to 24 h. Preproinsulin mRNA declined more slowly, reaching 35% of the control value at 6 h, and increased more rapidly, returning to the control value by 24 h. Pancreatic insulin levels did not decrease significantly until 24 h. Exposure of isolated rat islets to CPH for 30 min in vitro selectively inhibited proinsulin synthesis by 62%, without affecting preproinsulin mRNA levels. The dissociation between changes in proinsulin and preproinsulin mRNA levels suggests that the decrease in preproinsulin mRNA in vivo is associated with but does not cause CPH-induced changes in insulin biosynthesis.     

1,25-Dihydroxyvitamin D3 enhances cytosolic free calcium in HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) caused a rise in the concentration of intracellular free calcium ions ([Ca2+]i) in HL-60 cells. This effect of 1,25(OH)2D3 parallels its suppression of cell proliferation and its induction of cell differentiation into monocyte-like cells. The changes in [Ca2+]i are dose and time dependent. The concentration of 1,25(OH)2D3 (10(-7) M) that induced maximal differentiation also caused the maximal increase in intracellular Ca2+. The rise in cytoplasmic free Ca2+ concentration was not immediate and reached statistical significance only after 24 h. The [Ca2+]i reached its peak at 48 h (134 +/- 4 nM vs 101 +/- 3 nM in controls) and remained stable at this level. The increase in intracellular Ca2+ was found to be related to new protein synthesis, because it was inhibited in the presence of specific RNA and protein synthesis inhibitors. The rise in [Ca2+]i was not observed during incubation of HL-60 cells with 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), a vitamin D metabolite that does not induce the differentiation of HL-60 cells. In contrast, 25-hydroxyvitamin D3 (25-OH-D3) and phorbol 12-myristate 13-acetate (TPA), both of which induce differentiation in this cell line, also increase [Ca2+]i. In conclusion, the present study emphasizes that a significant increase in intracellular free Ca2+ occurs in the effect of 1,25(OH)2D3 on HL-60 cells.     

Differential regulation of steady state 4-ene steroid 5 alpha-reductase messenger ribonucleic acid levels along the rat epididymis

Epididymal nuclear 5 alpha-reductase enzyme activity is regulated by a testosterone-dependent factor from the testis. Regulation at the mRNA level, however, has not been investigated. Endocrine manipulation experiments were designed to determine whether 5 alpha-reductase is regulated at the steady state mRNA level. Steady state mRNA concentrations were assessed using the full-length cDNA for female rat liver 5 alpha-reductase. Longitudinal distribution showed that the highest mRNA concentrations were present in the initial segment of the caput epididymidis and were 3- to 7-fold higher than in the other tissue segments. The androgen dependence of the mRNA levels for 5 alpha-reductase was assessed by bilateral orchidectomy and simultaneous testosterone replacement therapy. One week after surgery, mRNA concentrations in orchidectomized rats were decreased to 15% of control levels in the initial segment of the caput epididymidis and to 40-50% of control levels in the remaining epididymal segments. Administration of testosterone at a dose that mimics normal serum concentrations (2.5-cm Silastic implant) restored 5 alpha-reductase mRNA concentrations to control levels in the corpus and cauda epididymidis, but these were not significantly different from orchidectomized levels (P greater than or equal to 0.05) in the initial segment and caput epididymidis. Administration of testosterone at a dose designed to approximate 5- to 8-fold normal serum concentrations (18.6-cm implant) maintained 5 alpha-reductase mRNA concentrations at only 50% of control levels in the initial segment, while complete maintenance was observed in the rest of the tissue. The effects of unilateral orchidectomy revealed that 5 alpha-reductase mRNA concentrations decrease selectively in the initial segment of the orchidectomized side. This is the first report that epididymal 5 alpha-reductase is regulated at the mRNA level and that the regulation is different with respect to the segment being studied.     

1,25-Dihydroxyvitamin D3 up-regulates the 1,25-dihydroxyvitamin D3 receptor in vivo.

The level of mRNA encoding the 1,25-dihydroxyvitamin D3 receptor in the intestine of vitamin D-deficient rats given 1,25-dihydroxyvitamin D3 was determined by Northern blot analysis using a 32P-labeled cDNA probe to the 1,25-dihydroxyvitamin D3 receptor. mRNA levels increased 10-fold above deficiency levels at 6 and 12 hr after an intravenous dose of 1,25-dihydroxyvitamin D3, returning to predosing levels at 24 hr. Total receptor protein level determined by an immunoradiometric assay was increased 2-fold at 12 hr. No change in unoccupied receptor levels determined by ligand-binding assay was observed during this period. These results suggest that 1,25-dihydroxyvitamin D3 increases receptor mRNA and total receptor level to maintain constant levels of unoccupied receptor.     

1,25-二羟维生素D3对慢性阻塞性肺疾病大鼠气道炎症的作用与机制

目的 研究1,25-二羟维生素D3[1,25(OH)2D3]对慢性阻塞性肺疾病(COPD)气道炎症的作用与机制.方法 雄性Wistar大鼠30只,分为对照组(A组)、COPD组(B组)和1,25(OH)2D3+COPD组(C组),每组10只,用熏香烟加气管注内毒素法建立大鼠COPD模型,在C组腹腔注射1,25(OH)2...     

1,25-Dihydroxyvitamin D3 enhances thyrotropin releasing hormone induced thyrotropin secretion in normal pituitary cells

The findings of specific binding of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in normal rat pituitary tissue and selective effects of 1,25-(OH)2D3 on gene expression in clonal pituitary tumour cells have suggested that vitamin D may regulate pituitary function. Therefore, the in vitro effect of 1,25-(OH)2D3 on normal pituitary cells was investigated. Primary anterior pituitary cell cultures prepared from female rats were maintained in experimental medium +/- 10(-8) M 1,25-(OH)2D3 for up to 24 h and then incubated with fresh experimental medium containing TRH (10(-10)-10(-8) M) or vehicle for 1 h. Pretreatment with 1,25-(OH)2D3 for 24 h led to increased TSH release at all TRH concentrations tested (P less than 0.0001), a decrease in the half-maximal stimulatory dose of TRH for TSH release from 2 X 10(-9) M to 0.4 X 10(-9) M, a 22% increase in maximal TSH release (P less than 0.01), and an 81% increase in TSH release at 10(-9) M TRH (P less than 0.001). 1 X 10(-9) M 1,25-(OH)2D3 increased TRH (10(-9) M)-induced TSH release by 20% (P less than 0.05) but 10(-7) M and 10(-6) M 25-hydroxyvitamin D3 (25-OH D3) had no effect. The effect of 1,25-(OH)2D3 on TRH (10(-9) M)-induced TSH release was evident within 8 h and was maximal by 16 h. There was no effect on basal TSH release, TSH accumulation in the medium in the preceding 24 h nor on cell-associated TSH. 1,25-(OH)2D3 pretreatment had no effect on TRH-induced PRL secretion, PRL accumulation in the medium nor on cell-associated PRL. We have shown that 1,25-(OH)2D3 acts selectively on the thyrotroph to enhance in vitro responsiveness to physiologically relevant concentrations of TRH. These findings are consistent with the reported autoradiographic localization of [3H]-1,25-(OH)2D3 in the thyrotroph and support a permissive or regulatory role of vitamin D in the normal pituitary gland.     

1,25-Dihydroxyvitamin D3 receptors identified in the rat heart

Specific receptors for 1,25-dihydroxyvitamin D3, the active hormonal form of vitamin D3, were demonstrated in low salt chromatin preparations from normal rat hearts. Sucrose gradient analysis of KCl-extracted chromatin yielded a significant (P less than 0.005) peak of specific [3H]1,25-dihydroxyvitamin D3 binding in the 3.6S region. The peak of [3H]1,25-dihydroxyvitamin D3 binding was abolished by excess 1,25-dihydroxyvitamin D3, but not by 50 nM 25-hydroxyvitamin D3 nor by 1.0 microM levels of estradiol-17B, cortisol, or promegestone, demonstrating steroid specificity characteristic for such receptors. Upon Scatchard analysis this putative cardiac 1,25-dihydroxyvitamin D3 receptor yielded a single binding component with high affinity (KD = 0.36 nM) and low capacity (Nmax = 33 fmol/g tissue). Coupled with evidence for the presence of calcium binding proteins in this tissue, these observations suggest functional roles for 1,25-dihydroxyvitamin D3 and its receptors in cardiac muscle, possibly in regulating intracellular effects of calcium.     

1,25-Dihydroxyvitamin D3 binding in estrogen-responsive rat breast tumor

Receptors for 1,25 dihydroxyvitamin D3 [1,25-(OH)2D3] have been reported in breast tissue; however, the presence of multiple binding sites and limited availability of human tumor tissue have precluded complete biochemical characterization of the receptor in breast cancer. In the present study, binding proteins for 1,25-(OH)2D3 in breast tumor tissue were analyzed using a rat model of breast cancer. Breast tumors were induced in adult female rats with the carcinogen nitrosomethylurea. Such tumors previously have been shown to possess high levels of estrogen receptors and are estrogen dependent. Binding proteins for 1,25-(OH)2D3 in 0.3 M KCl extracts of tumor tissue were analyzed on sucrose density gradients. Two binding proteins were detected: one sedimenting at 5-6 S representing binding of 1,25-(OH)2D3 to the 25 hydroxyvitamin D3 (25OHD3) binding protein and a second moiety sedimenting, like the rat intestinal receptor, at 3.3 S. Binding of the dihydroxy metabolite to the faster sedimenting protein could be eliminated by inclusion of radioinert 25OHD3 in the incubation medium. Receptor content of rat breast tumor was investigated using an hydroxylapatite assay by incubating tumor extracts with a saturating concentration of 1,25-(OH)2-[3H]D3, plus unlabeled 25OHD3 to eliminate binding of the hormone to the 5-6 S species. Scatchard analysis of 1,25-(OH)2D3 binding to the tumor extracts yielded an apparent dissociation constant (Kd) of 0.33 nM. In summary, breast tumors induced in rats by nitrosomethylurea were shown to contain high affinity 1,25-(OH)2D3 receptors with properties very similar to those reported for the receptor in other mammalian target organs. The presence of receptors for 1,25-(OH)2D3 in these rat breast tumors implies that the tissue is potentially responsive to the hormone.     

1,25-二羟维生素D3对支气管哮喘大鼠气道炎症和肺内诱导型一氧化氮合酶的影响

目的本文探讨1,25-二羟维生素D3[1,25-dihydroxy vitmin D3,1,25-(OH)2D3]对于支气管哮喘(简称哮喘)大鼠气道炎症和肺内诱导型一氧化氮合酶(induced nitric oxide synthase,iNOS)的影响。方法50只Wistar大鼠随机分为四组:正常对照组,哮喘组,预防处理组和治疗组。用卵蛋白作为致敏原制备哮喘大鼠模型,第一组预防处理组在致敏前三天给予口服1,25-(OH)2D3,共给药3周,第二组预防组则于致敏第1天和第7天给予肌注维生素D3。治疗组则于支气管激发开始前给药处理。应用动物肺功能仪测定大鼠对于乙酰甲胆碱刺激的气道反应性,测定支气管肺泡灌洗液(BALF)细胞总数及嗜酸粒细胞计数和肺组织病理变化。检测肺组织中一氧化氮(NO)含量,iNOS活性和iNOS mRNA表达水平,用免疫组织化学染色方法检测肺组织iNOS的表达并观察iNOS在气道组织分布的改变。结果1,25-(OH)2D3预防及治疗组可减轻BALF中细胞总数及嗜酸粒细胞计数,同时降低肺组织中NO含量、iNOS活性及大鼠肺组织中iNOS mRNA的表达水平。免疫组织化学结果显示,干预组可降低哮喘大鼠气道组织中上皮细胞和巨嗜细胞中iNOS阳性。结论1,25-(OH)2D3可降低哮喘大鼠气道炎症及iNOS表达水平,提示1,25-(OH)2D3对于哮喘可能有潜在的治疗作用。     

Follistatin steady state messenger ribonucleic acid levels in confluent cultures of a rat renal mesangial cell line are regulated by multiple factors.

The regulation of steady state levels of follistatin (FS) messenger RNA (mRNA) was examined in a rat renal mesangial cell line in tissue culture. A specific 32P-radiolabeled antisense probe was used which corresponds to the 3' end of exon 5 together with the 5' end of exon 6 of the rat FS gene, and which distinguishes between the two different forms of FS mRNA. In addition, a specific 35S-radiolabeled probe for the ubiquitous protein cyclophilin was developed and used as an internal standard. Total RNA was harvested from confluent cell cultures to yield four independent samples per treatment/time point, and equal amounts of RNA from every sample in a given experiment were subjected to S1-nuclease analysis for the estimation of specific mRNA levels. Treatment of the cultured cells with epidermal growth factor (10 nM) caused an 8- to 9-fold increase in the FS mRNA level after 4 h, but no consistent change was observed after treatment with basic fibroblast growth factor (0.28 or 0.56 nM), somatostatin (3.7-73 nM), angiotensin II (0.1-2500 nM), or FS itself (0.29 nM) for between 4 and 48 h. Neither activin (0.5 or 1.2 nM) nor inhibin (0.64 nM) changed the FS mRNA level in the mesangial cell line during a 24-h treatment. FS mRNA levels in the cells also were not affected by a 48-h treatment with the steroids dihydrotestosterone (1-1000 nM), estradiol (1 and 100 nM), and the antiprogesterone RU 486 (1000 nM), whereas 100 nM RU 28362 (a synthetic glucocorticoid) caused a 5- to 6-fold increase and 1000 nM progesterone increased the FS mRNA level up to 3.5-fold above control. Retinoic acid, a vitamin A derivative, significantly increased the FS steady state mRNA level at 3 nM, and at 1000 nM stimulated FS mRNA up to 5-fold within 4 h, whereas incubation of the cells with 30 microM prostaglandin E2 for 4 h caused a 10-fold increase. The FS mRNA level increased 3- and 4-fold within 4 h during incubation of the cells with 100 nM phorbol 12-myristate, 13-acetate, and 25 microM forskolin, respectively, whereas the calcium ionophore A23187 (1-100 microM) caused no change within this timespan. None of the tested hormones had an obvious effect on the ratio of the two different forms of FS mRNA (FS 344:FS 317).(ABSTRACT TRUNCATED AT 400 WORDS)     

1,25-Dihydroxyvitamin D3 in the treatment of idiopathic thrombocythemia and myelofibrosis

The effect of treatment with 1,25-dihydroxyvitamin D3 administered at the dose of 1.50-3.00 ug/day for at least 12 months was evaluated in three patients with idiopathic myelofibrosis and in five patients with idiopathic thrombocythemia. This treatment did not cause any significant change in the hematological values or the clinical course of the myeloproliferative diseases in any of the patients. Based on these data, treatment with 1,25-dihydroxyvitamin D3 in non toxic doses seems to be of doubtful benefit in patients with these disorders.     

Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism

Vitamin D, via its receptor (VDR), inhibits the hormone secretion and proliferation of parathyroid cells. Vitamin D deficiency and reduced parathyroid VDR expression has been associated with development of hyperparathyroidism (HPT) secondary to uremia. VDR polymorphisms may influence VDR messenger RNA (mRNA) levels and have been coupled to an increased risk of parathyroid adenoma of primary HPT. VDR mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA levels were determined by RNase protection assay in 42 single parathyroid adenomas of patients with primary HPT, 23 hyperplastic glands of eight patients with uremic HPT, and 15 normal human parathyroid glands. The adenomas and hyperplasias demonstrated similar VDR mRNA levels, which were reduced (42 +/- 2.8% and 44 +/- 4.0%) compared with the normal glands (P < 0.0001). Comparison of parathyroid adenoma with a normal-sized parathyroid gland of the same individual (n = 3 pairs) showed a 20-58% reduction in the tumor. Nodularly enlarged glands represent a more advanced form of secondary HPT and showed greater reduction in the VDR mRNA levels than the diffusely enlarged glands (P < 0.005). The reduced VDR expression is likely to impair the 1,25(OH)2D3-mediated control of parathyroid functions, and to be of importance for the pathogenesis of not only uremic but also primary HPT. Circulating factors like calcium, PTH, and 1,25(OH)2D3 seem to be less likely candidates mediating the decreased VDR gene expression in HPT.     

1,25-Dihydroxyvitamin D and vitamin D-binding protein are both decreased in streptozotocin-diabetic rats

Calcium and vitamin D metabolism were studied in streptozotocin-treated rats up to 10 days after the induction of diabetes. Proteinuria, hypercalciuria, and hyperphosphaturia appeared as early as 3 days after diabetes induction and were reversed by insulin. The serum proteins and fasting calcium concentrations were decreased in untreated diabetic rats. The concentration of serum vitamin D binding protein (DBP) was higher in male than in female control rats (mean +/- SD; 555 +/- 73 vs. 348 +/- 28 mg/liter, P less than 0.001). When sequentially measured in male untreated diabetic rats, DBP concentration steadily decreased. Compared with control values, DBP was reduced 19%, 28%, and 32% on days 3, 6, and 10, respectively, after induction of diabetes in male rats. In female animals, DBP was reduced 22% on day 10 of diabetes. DBP concentration was corrected by insulin treatment of diabetic rats and remained normal in streptozotocin-treated animals that did not develop diabetes. The serum concentration of 25-hydroxyvitamin D3 was similar in both sexes and was not affected by diabetes. Like DBP, the concentration of total 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was higher in male than in female control rats (120 +/- 24 vs. 96 +/- 17 ng/liter, P less than 0.001), but 10 days after induction of diabetes this concentration decreased by 37% and 29% in male and female rats, respectively. The free 1,25-(OH)2D3 concentration, estimated from the molar 1,25-(OH)2D3/DBP ratio, was similar in both sexes and was not decreased by diabetes. We conclude that experimental diabetes in the rat induces a decrease in DBP concentration and a concomitant decrease in total but not in free 1,25-(OH)2D3 concentrations. This may indicate that diabetes decreases circulating 1,25-(OH)2D3 concentrations through alterations in DBP levels.     

The effect of thyroid hormone on fibronectin messenger ribonucleic acid levels in primary cultured rat hepatocytes.

Our previous in vivo studies demonstrated that thyroid hormone promotes the expression of the fibronectin (FN) gene in the rat liver, while it inhibits the synthesis in cultured human skin fibroblasts. These results can be interpreted as either different regulation of FN synthesis or gene expression among tissues, or divergent results of experiments performed in vivo or in vitro. Here we report on the action of thyroid hormone on FN gene expression in vitro using primary cultured hepatocytes compared to that in cultured skin fibroblasts. Hepatocytes were isolated from hypothyroid rats and were cultured in medium supplemented with thyroidectomized bovine serum (TxBS) or fetal bovine serum (FBS). T3 was added 2 or 24 h after plating, and cells were harvested after 2, 6, or 24 h. Total RNA was extracted, and mRNAs for rat FN and albumin were measured. The requirement of de novo protein synthesis for thyroid hormone-mediated induction of FN mRNA was examined by the addition of cycloheximide 15 min before T3 addition. The amount of FN mRNA significantly decreased in the hepatocytes cultured with TxBS compared with those cultured with FBS. The addition of T3 to TxBS resulted in the restoration of FN mRNA to the level in hepatocytes cultured in FBS. FN mRNA increased during the course of culture in the absence of T3; however, a further increase was observed 6 h after T3 addition. The abundance of albumin RNA decreased during the course of culture, but unlike FN mRNA, it was not changed by T3 addition. The increase in FN mRNA by T3 was not influenced by cycloheximide. These results indicate that thyroid hormone enhances FN gene expression in hepatocytes by its direct action without requiring de novo protein synthesis. In contrast, T3 decreased FN mRNA in cultured skin fibroblasts. Thus, the mode of thyroid hormone action on FN gene expression is different among tissues.